CN110646391A - Mitochondrial fluorescence labeling method for angiosperm flower organs - Google Patents
Mitochondrial fluorescence labeling method for angiosperm flower organs Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 41
- 210000000056 organ Anatomy 0.000 title claims abstract description 18
- 241000218922 Magnoliophyta Species 0.000 title claims abstract description 16
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 11
- 238000001215 fluorescent labelling Methods 0.000 title claims abstract description 11
- 230000002438 mitochondrial effect Effects 0.000 title description 5
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 60
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 40
- 241000196324 Embryophyta Species 0.000 claims abstract description 19
- 239000003085 diluting agent Substances 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 14
- 239000013543 active substance Substances 0.000 claims abstract description 9
- 239000012895 dilution Substances 0.000 claims abstract description 7
- 238000010790 dilution Methods 0.000 claims abstract description 7
- 238000010438 heat treatment Methods 0.000 claims abstract description 7
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 6
- 238000004113 cell culture Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000000975 dye Substances 0.000 claims 1
- 239000001046 green dye Substances 0.000 abstract description 6
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 abstract description 2
- 108050008072 Cytochrome c oxidase subunit IV Proteins 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000003463 organelle Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000224432 Entamoeba histolytica Species 0.000 description 1
- 241000224467 Giardia intestinalis Species 0.000 description 1
- 241000243190 Microsporidia Species 0.000 description 1
- 230000004103 aerobic respiration Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 229940007078 entamoeba histolytica Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940085435 giardia lamblia Drugs 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- General Physics & Mathematics (AREA)
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a fluorescence labeling method for mitochondria of angiosperm floral organs, belonging to the technical field of biological research, which comprises the following steps: the method comprises the following steps: adding a fluorescent agent for marking into a diluent for dilution; step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent; step three: making plant flower organs into specimens; step four: dripping the fluorescent agent on the specimen; step five: observing the state through a microscope, wherein the diluent in the first step is physiological saline, the ratio of the physiological saline to the fluorescent agent is 100:1, the fluorescent agent is Jianna green dye solution, and the surface activity of the fluorescent agent is improved by adding an active agent into the fluorescent agent, so that the fluorescent agent can be quickly oxidized with cytochrome oxidase in mitochondria when being dripped onto an experimental specimen, and the fluorescence labeling speed is improved.
Description
Technical Field
The invention relates to the technical field of biological research, in particular to a mitochondrial fluorescence labeling method for angiosperm flower organs.
Background
Angiosperms are the most evolved, most diverse, most widely distributed and most adaptable group in the world plant kingdom today. It is known that the angiosperms account for more than 20 ten thousand species in total and account for more than half of the total number of plant kingdoms all over the world. The known angiosperm in China belong to more than 2700 genera and more than 3 ten thousand species. The angiosperm and human being have close relationship, for example, the angiosperm in China can provide more than 2000 kinds of food; more than 300 fruit trees are available; the number of the flower plants is not enough; the medicinal angiosperm plants comprise 10027 (including the following classification units) which account for 90 percent of the total number of medicinal plants in China and are the group with the most medicinal types, and most of the traditional Chinese medicines are from the angiosperm plants.
Mitochondria, a two-layer membrane-coated organelle present in most cells, are structures that produce energy in cells, are the main sites for aerobic respiration of cells, and are called "powerhouse". The diameter is about 0.5 to 1.0 micron.
Most eukaryotic cells, except entamoeba histolytica, giardia lamblia and several microsporidia, possess more or less mitochondria, but their individual mitochondria differ in size, number and appearance.
Mitochondria possess their own genetic material and genetic system, but have a limited genome size and are a semi-autonomous organelle. In addition to powering cells, mitochondria are involved in processes such as cell differentiation, cell information transmission, and apoptosis, and possess the ability to regulate cell growth and cell cycle.
At present, when mitochondria are researched, the positions of the mitochondria need to be marked through fluorescence, so that the moving track of the mitochondria can be observed through a microscope, but the fluorescence is between the mitochondria marking, a user needs to inject a fluorescent agent into flower organs of plants, then waits for the fluorescent agent to be contacted with the mitochondria, and the fluorescent agent cannot be attracted by the mitochondria due to the characteristics of the fluorescent agent and needs to freely fall onto the mitochondria, so that the waiting time of the step is longer, and the whole experiment time is increased.
Disclosure of Invention
The invention aims to provide a mitochondrial fluorescence labeling method for angiosperm floral organs, which aims to solve the problem that the experiment process is too long due to the slow reaction time of a fluorescent agent and mitochondria in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a mitochondrial fluorescence labeling method of angiosperm floral organs comprises the following steps:
the method comprises the following steps: adding a fluorescent agent for marking into a diluent for dilution;
step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;
step three: making plant flower organs into specimens;
step four: dripping the fluorescent agent on the specimen;
step five: the state was observed by a microscope.
Preferably, the diluent in the first step is physiological saline, the ratio of the physiological saline to the fluorescent agent is 100:1, and the fluorescent agent is the Jianna green dye solution.
Preferably, the temperature for raising the temperature of the fluorescent agent in the second step is 30-40 ℃.
Preferably, the preparation method of the specimen in the third step is that the position of the separated plant flower part is obliquely cut downwards into a 3-5mm thin slice by using a blade, the bottom of the thin slice is placed in a laboratory dish, then the cell culture solution is added, and the thin slice is stood for 24 hours.
Preferably, the fourth specific operation step is to drop the diluted fluorescent agent onto a sheet in a laboratory dish through a dropper, and standing for 10-15 min.
Preferably, the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to a state in which the mitochondria can be clearly observed.
Compared with the prior art, the invention has the beneficial effects that: by the fluorescent marking method for the mitochondria of the flower organs of angiosperm, at present, when the mitochondria are researched, the position of the mitochondria needs to be marked by fluorescence, so that the moving track of the mitochondria can be observed through a microscope, but the fluorescence is between the mitochondria markers, a user needs to inject the fluorescent agent into the flower organ of the plant and then wait for the fluorescent agent to contact the mitochondria, because the fluorescent agent is not attracted by mitochondria due to the characteristics of the fluorescent agent, the fluorescent agent is required to fall onto the mitochondria freely, the waiting time of the step is longer, the whole experiment time is increased, in the application document, the surface activity of the fluorescent agent is improved by adding the active agent into the fluorescent agent, so that the fluorescent agent can be quickly oxidized with cytochrome oxidase in mitochondria when being dripped onto an experimental specimen, and the fluorescence labeling speed is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides the following technical scheme: a fluorescence labeling method for mitochondria of angiosperm floral organs is used for increasing fluorescence labeling speed and reducing experiment time.
Example 1
The marking method comprises the following steps:
the method comprises the following steps: 0.5g of fluorescent agent for marking is added into 50ml of diluent for dilution;
step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;
step three: making plant flower organs into specimens;
step four: dripping the fluorescent agent on the specimen;
step five: the state was observed by a microscope.
Wherein, the diluent in the first step is normal saline, and the fluorescent agent is a Jianna green dye solution.
Wherein the temperature of the fluorescent agent in the second step is 30-40 ℃.
The preparation method of the specimen in the third step comprises the steps of obliquely downwards cutting the separated flower part of the plant into slices of 3mm by using a blade, placing the bottom of the slices in an experimental dish, adding cell culture solution, and standing for 24 hours.
And the fourth specific operation step is to drip the diluted fluorescent agent onto a sheet in a laboratory dish through a dropper, and standing for 10-15 min.
And the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to the state that the mitochondria can be clearly observed.
Example 2
The marking method comprises the following steps:
the method comprises the following steps: 0.5g of fluorescent agent for marking is added into 50ml of diluent for dilution;
step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;
step three: making plant flower organs into specimens;
step four: dripping the fluorescent agent on the specimen;
step five: the state was observed by a microscope.
Wherein, the diluent in the first step is normal saline, and the fluorescent agent is a Jianna green dye solution.
Wherein the temperature of the fluorescent agent in the second step is 30-40 ℃.
The preparation method of the specimen in the third step comprises the steps of obliquely downwards cutting the separated flower part of the plant into 4mm thin slices by using a blade, placing the bottom of the thin slices in an experimental dish, adding cell culture solution, and standing for 24 hours.
And the fourth specific operation step is to drip the diluted fluorescent agent onto a sheet in a laboratory dish through a dropper, and standing for 10-15 min.
And the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to the state that the mitochondria can be clearly observed.
Example 3
The marking method comprises the following steps:
the method comprises the following steps: 0.5g of fluorescent agent for marking is added into 50ml of diluent for dilution;
step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;
step three: making plant flower organs into specimens;
step four: dripping the fluorescent agent on the specimen;
step five: the state was observed by a microscope.
Wherein, the diluent in the first step is normal saline, and the fluorescent agent is a Jianna green dye solution.
Wherein the temperature of the fluorescent agent in the second step is 30-40 ℃.
The preparation method of the specimen in the third step comprises the steps of obliquely downwards cutting the separated flower part of the plant into 5mm thin slices by using a blade, placing the bottom of the thin slices in an experimental dish, adding cell culture solution, and standing for 24 hours.
And the fourth specific operation step is to drip the diluted fluorescent agent onto a sheet in a laboratory dish through a dropper, and standing for 10-15 min.
And the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to the state that the mitochondria can be clearly observed.
Example 4
The marking method comprises the following steps:
the method comprises the following steps: 0.5g of fluorescent agent for marking is added into 50ml of diluent for dilution;
step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;
step three: making plant flower organs into specimens;
step four: dripping the fluorescent agent on the specimen;
step five: the state was observed by a microscope.
Wherein, the diluent in the first step is normal saline, and the fluorescent agent is a Jianna green dye solution.
Wherein the temperature of the fluorescent agent in the second step is 30-40 ℃.
The preparation method of the specimen in the third step comprises the steps of obliquely downwards cutting the separated flower part of the plant into 5mm thin slices by using a blade, placing the bottom of the thin slices in an experimental dish, adding cell culture solution, and standing for 20 hours.
And the fourth specific operation step is to drip the diluted fluorescent agent onto a sheet in a laboratory dish through a dropper, and standing for 10-15 min.
And the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to the state that the mitochondria can be clearly observed.
In the above examples, the lines and moving directions generated after the mitochondria are marked are clearly observed by the fluorescent agent through a microscope, and the marking state of each group is recorded, and the results are as follows:
compared with the existing marking method, the marking method in the application document can effectively improve the marking speed of mitochondria and reduce the experiment time.
While the invention has been described with reference to certain embodiments, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the various embodiments of the invention disclosed may be used in any combination whatsoever without structural conflict, and the failure to exhaustively describe such combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (6)
1. A fluorescence labeling method for mitochondria of angiosperm floral organs is characterized in that: the marking method comprises the following steps:
the method comprises the following steps: adding a fluorescent agent for marking into a diluent for dilution;
step two: heating and dissolving the diluted fluorescent agent, and then adding an active agent;
step three: making plant flower organs into specimens;
step four: dripping the fluorescent agent on the specimen;
step five: the state was observed by a microscope.
2. The method of claim 1, wherein the method comprises the steps of: in the first step, the diluent is physiological saline, the ratio of the physiological saline to the fluorescent agent is 100:1, and the fluorescent agent is the Jiannalv dye solution.
3. The method of claim 1, wherein the method comprises the steps of: and the temperature for raising the temperature of the fluorescent agent in the second step is 30-40 ℃.
4. The method of claim 1, wherein the method comprises the steps of: the preparation method of the specimen in the third step comprises the steps of obliquely downwards cutting the separated flower part of the plant into 3-5mm thin slices by using a blade, placing the bottom of the thin slices in an experimental dish, adding cell culture solution, and standing for 24 hours.
5. The method of claim 1, wherein the method comprises the steps of: the fourth specific operation step is to drop the diluted fluorescent agent onto a thin sheet in a laboratory dish through a dropper, and standing for 10-15 min.
6. The method of claim 1, wherein the method comprises the steps of: and the observation method in the fifth step is to record the fluorescence label of the mitochondria at the same position after the microscope is adjusted to the state that the mitochondria can be clearly observed.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103788940A (en) * | 2012-11-02 | 2014-05-14 | 国家纳米科学中心 | Aggregation-induced emission fluorescent molecule as well as preparation method and fluorescent dye composition, and application of aggregation-induced emission fluorescent molecule and fluorescent dye composition in mitochondria dyeing |
| CN103983496A (en) * | 2014-05-19 | 2014-08-13 | 北京林业大学 | Mitochondrial fluorescence labeling method for angiosperm flower organs |
| CN105717083A (en) * | 2016-02-02 | 2016-06-29 | 内蒙古农业大学 | Intelligent fluorescent label and preparation method and application thereof |
| US20160299078A1 (en) * | 2013-12-03 | 2016-10-13 | The Hong Kong University Of Science And Technology | Specific Detection and Quantification of Cardiolipin and Isolated Mitochondria by Positively Charged AIE Fluorogens and Method of Manufacturing Thereof |
| CN107290197A (en) * | 2017-06-26 | 2017-10-24 | 四川省人民医院 | A kind of living cells mitochondria quantity dyes detection method |
-
2019
- 2019-09-27 CN CN201910927229.2A patent/CN110646391A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103788940A (en) * | 2012-11-02 | 2014-05-14 | 国家纳米科学中心 | Aggregation-induced emission fluorescent molecule as well as preparation method and fluorescent dye composition, and application of aggregation-induced emission fluorescent molecule and fluorescent dye composition in mitochondria dyeing |
| US20160299078A1 (en) * | 2013-12-03 | 2016-10-13 | The Hong Kong University Of Science And Technology | Specific Detection and Quantification of Cardiolipin and Isolated Mitochondria by Positively Charged AIE Fluorogens and Method of Manufacturing Thereof |
| CN103983496A (en) * | 2014-05-19 | 2014-08-13 | 北京林业大学 | Mitochondrial fluorescence labeling method for angiosperm flower organs |
| CN105717083A (en) * | 2016-02-02 | 2016-06-29 | 内蒙古农业大学 | Intelligent fluorescent label and preparation method and application thereof |
| CN107290197A (en) * | 2017-06-26 | 2017-10-24 | 四川省人民医院 | A kind of living cells mitochondria quantity dyes detection method |
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| 《实验指导与实验报告》编写组: "《实验指导与实验报告 生物 高中必修1 人教版》", 31 July 2016, 湖南教育出版社 * |
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Application publication date: 20200103 |